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. 2014 Oct;80(20):6334–6345. doi: 10.1128/AEM.01573-14

Two Novel Toxin Variants Revealed by Whole-Genome Sequencing of 175 Clostridium botulinum Type E Strains

K A Weedmark a, D L Lambert b,*, P Mabon a, K L Hayden a, C J Urfano a, D Leclair b,*, G Van Domselaar a, J W Austin b, C R Corbett a,✉
Editor: H Nojiri
PMCID: PMC4178653  PMID: 25107978

Abstract

We sequenced 175 Clostridium botulinum type E strains isolated from food, clinical, and environmental sources from northern Canada and analyzed their botulinum neurotoxin (bont) coding sequences (CDSs). In addition to bont/E1 and bont/E3 variant types, neurotoxin sequence analysis identified two novel BoNT type E variants termed E10 and E11. Strains producing type E10 were found along the eastern coastlines of Hudson Bay and the shores of Ungava Bay, while strains producing type E11 were only found in the Koksoak River region of Nunavik. Strains producing BoNT/E3 were widespread throughout northern Canada, with the exception of the coast of eastern Hudson Bay.

INTRODUCTION

Botulism, a rare and severe disease characterized by a descending flaccid paralysis, is caused by botulinum neurotoxin (BoNT), the most potent toxin known. BoNT is produced by phylogenetically distinct anaerobic spore-forming bacteria grouped under the taxonomic designation of Clostridium botulinum. Rare botulinogenic strains of related clostridia, such as C. baratii, C. butyricum, and C. argentinense, have also been observed (1, 2). Seven serologically distinct BoNTs (A to G) can be distinguished based on neutralization of toxicity with specific antisera. Recently, a strain of C. botulinum producing botulinum neurotoxin type B (BoNT/B) and another BoNT that is not neutralized by antitoxins to BoNTs A to G has been isolated from a case of infant botulism (3). It has been proposed that this novel neurotoxin is an eighth serotype, BoNT/H (3, 4). The botulinum neurotoxins are comprised of three structural domains (translocation [HN], receptor binding [HC], and catalytic [LC]). These toxins target different SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor) proteins in the neuromuscular junction to block neurotransmitter release (5, 6).

Heterogeneity within the BoNTs has led to a classification of BoNTs into subtypes. Within a toxin serotype, differences in amino acid sequences can range from 0.9% to 25% (2, 7). Hill and Smith (8) point out that the newer subtypes have been defined based on their DNA sequence and propose the use of the term “subtype/genetic variant” to avoid confusion with the historical use of “subtype,” utilized to designate immunological or enzymatic differences among neurotoxins. Two approaches have been used to define new BoNT variants. The first uses a cutoff value of 2.5% difference in amino acid composition (9–11), whereas the second relies on a phylogenetic approach in which variants correspond to clades formed by the clustering of bont sequences (1, 7, 12) Based on these methods, bont/A1 to bont/A5, bont/B1 to bont/B7, bont/E1 to bont/E9, and bont/F1 to bont/F7 gene variants have been described (1, 2, 8, 10, 12–17).

In Canada, C. botulinum type E is the predominant BoNT serotype associated with food-borne botulism, accounting for 86.2% of all laboratory-confirmed food-borne botulism outbreaks reported between 1985 and 2005 (18). Although variant types E1, E3, and E7 have been identified in three separate Canadian isolates (2), little is known about BoNT/E variants involved in the majority of Canadian outbreaks.

In this study, the bont/E variant coding sequences (CDSs) for 175 C. botulinum type E isolates derived from food, clinical, and environmental sources were obtained using shotgun next-generation sequencing and compared to previously determined bont/E variants. Here, we present phylogenetic and sequence identity analyses of bont/E variants and report on the identification of two new variants, types E10 and E11. We also describe the geographic distribution of strains carrying these bont/E variants and the involvement of these strains in food-borne botulism incidents.

MATERIALS AND METHODS

Culture conditions, DNA isolation, and genome sequencing.

A total of 175 C. botulinum group II strains were cultured at room temperature for 48 to 72 h in an atmosphere of 10% H2, 10% CO2, and 80% N2 using 1.5% McClung-Toabe agar (Difco, Tucker, GA), supplemented with 5% egg yolk extract, and 5% yeast extract (Difco). Single colonies were inoculated into 10 ml of TPGY (5% [wt/vol] tryptone [Difco], 0.5% [wt/vol] peptone [Difco], 0.4% [wt/vol] glucose [Difco], 2% [wt/vol] yeast extract [Difco], and 0.1% sodium thioglycolate [Sigma, St. Louis, MO]) medium for 24 h. Genomic DNA from C. botulinum strains listed in Table 1 was extracted using the Qiagen DNeasy blood and tissue kit (Qiagen, Mississauga, Canada), and 16S rRNA sequencing was performed to verify homology to C. botulinum group II species (data not shown). Paired-end libraries were prepared using the Nextera or TruSeq kit according to the manufacturer's instructions (Illumina Inc., San Diego, CA). Library insert lengths (range, 200 bp to 1,000 bp) were estimated using an Agilent bioanalyzer (Agilent, Mississauga, Ontario, Canada). Sequencing was performed using paired-end sequencing by synthesis (run parameters: 2 × 25 to 2 × 250 cycles) on a GAIIx or MiSeq instrument according to the manufacturer protocols (Illumina). Reads were trimmed (Phred score < 25; trim ≤ 5 nucleotides [nt]) and filtered to include reads ≥25 nt long with a q-score of ≥30 (CG-Pipeline r432, http://sourceforge.net/projects/cg-pipeline). Average read coverage for all isolates exceeded 50-fold based on an average 3.8-Mb genome.

TABLE 1.

Clostridium botulinum group II BoNT/E strains studieda

Isolate OB BoNT Year Sample type Origin Region Location Source
211 VH Dolman 1 E3 1949 Clinical Pickled herring PAC Vancouver, BC NML (35, 36)
E1 Dolman 2 E1 <1980 Clinical ND PAC ND NML (35)
E-RUSS 3 E1 ∼1936 Clinical Sturgeon intestine UKR Sea of Azov, Ukraine BRS (30, 36–39)
MU9708EJG-F235 15 E3 1997 Clinical Muktuk NWT Aklavik, NT BRS
MU9708EJG-F236 15 E3 1997 Clinical Muktuk NWT Aklavik, NT BRS
MU0103EMS 24 E3 2001 Clinical Muktuk oil NWT Aklavik, NT BRS
FE9909ERG 22 E3 1999 Clinical Feces NWT Inuvik, NT BRS (37, 38)
FE0005EJT 23 E3 2000 Clinical Feces NWT Inuvik, NT BRS (37, 38)
MU0005EJT 23 E3 2000 Clinical Muktuk NWT Inuvik, NT BRS (37, 38)
MU8903E 6 E3 1989 Clinical Muktuk NWT Paulatuk, NT BRS
SO301E E10 2001 Environmental Shoreline soil EHB Kuujjuaraapik, QC BRS (27)
SO303E1 E10 2001 Environmental Shoreline soil EHB Umiujaq, QC BRS (27)
SO303E3 E10 2001 Environmental Shoreline soil EHB Umiujaq, QC BRS (27)
SO303E4 E10 2001 Environmental Shoreline soil EHB Umiujaq, QC BRS (27)
SO303E5 E10 2001 Environmental Shoreline soil EHB Umiujaq, QC BRS (27)
SO304E1 E10 2003 Environmental Shoreline soil EHB Inukjuak, QC BRS (27)
SO304E2 E10 2003 Environmental Shoreline soil EHB Inukjuak, QC BRS (27)
SO305E1 E10 2003 Environmental Shoreline soil EHB Inukjuak, QC BRS (27)
SO305E2 E10 2003 Environmental Shoreline soil EHB Inukjuak, QC BRS(27)
SO307E1 E10 2003 Environmental Shoreline soil EHB Puvirnituq, QC BRS (27)
SO309E2 E10 2003 Environmental Shoreline soil EHB Puvirnituq, QC BRS (27, 37, 38)
SP417E-Alc E10 2001 Environmental Coastal rock EHB Puvirnituq, QC BRS (27)
SP417E-NT E10 2001 Environmental Coastal rock EHB Puvirnituq, QC BRS (27)
BE9708E1 14 E3 1997 Clinical Beluga WHB Arviat, NU BRS
CA9708E1 14 E3 1997 Clinical Caribou WHB Arviat, NU BRS
FE9708E1JI 14 E3 1997 Clinical Feces WHB Arviat, NU BRS
FE9708E1PI 14 E3 1997 Clinical Feces WHB Arviat, NU BRS
GA0811E1IT 30 E3 2008 Clinical Gastric liquid BI Baffin Island, NU BRS
FE0801E1IT 30 E3 2008 Clinical Feces BI Kimmirut, NU BRS
Bennett 5 E3 1976 Clinical Gastric liquid LAB Happy Valley, NL BRS (37, 38)
FE9908EDL 21 E3 1999 Clinical Feces UB Aupaluk, QC BRS
FE9507EEA 7 E3 1995 Clinical Feces UB Kangiqsualujjuaq, QC BRS (37, 38)
MI9507E 7 E3 1995 Clinical Seal misiraq UB Kangiqsualujjuaq, QC BRS (37, 38)
FE9709EBB 17 E3 1997 Clinical Feces UB Kangiqsualujjuaq, QC BRS (37, 38)
FE9709ECB 17 E3 1997 Clinical Feces UB Kangiqsualujjuaq, QC BRS
FE9709ELB 17 E3 1997 Clinical Feces UB Kangiqsualujjuaq, QC BRS (37, 38)
GA9709EHS 17 E3 1997 Clinical Gastric liquid UB Kangiqsualujjuaq, QC BRS (37, 38)
GA9709EJA 17 E3 1997 Clinical Gastric liquid UB Kangiqsualujjuaq, QC BRS (37, 38)
GA9709ENS 17 E3 1997 Clinical Gastric liquid UB Kangiqsualujjuaq, QC BRS (37, 38)
FE9709EBB2 18 E10 1997 Clinical Feces UB Kangiqsualujjuaq, QC BRS
FE9709EKA 18 E10 1997 Clinical Feces UB Kangiqsualujjuaq, QC BRS
MI19709E 18 E10 1997 Clinical Seal igunaq UB Kangiqsualujjuaq, QC BRS
MI59709E 18 E10 1997 Clinical Seal igunaq UB Kangiqsualujjuaq, QC BRS
MI69709E 18 E10 1997 Clinical Seal igunaq UB Kangiqsualujjuaq, QC BRS
INGR16-02E1 E3 2002 Marine mammal Seal intestine UB Kangiqsualujjuaq, QC BRS
INWB2202E1 E3 2002 Marine mammal Seal intestine UB Kangiqsujuaq, QC BRS
SO321E1 E3 2001 Environmental Shoreline soil UB Kangirsuk, QC BRS (27)
SO322E1 E10 2001 Environmental Shoreline soil UB Kangirsuk, QC BRS (27)
Gordon 4 E3 1975 Clinical Clinical specimen UB Kuujjuaq, QC BRS (37, 38)
F9508EMA 8 E3 1995 Clinical Feces UB Kuujjuaq, QC BRS (37, 38)
VI9508E 8 E3 1995 Clinical Seal igunaq UB Kuujjuaq, QC BRS (37, 38)
S9510E 10 E3 1995 Clinical Seal meat UB Kuujjuaq, QC BRS (37, 38)
GA9811E1MS 19 E3 1998 Clinical Gastric liquid UB Kuujjuaq, QC BRS
GA9811E2MS 19 E3 1998 Clinical Gastric liquid UB Kuujjuaq, QC BRS
V9804E 20 E3 1998 Clinical Seal meat UB Kuujjuaq, QC BRS
FE0211E1DK 27 E3 2002 Clinical Feces UB Kuujjuaq, QC BRS
GA0702E1 29 E3 2007 Clinical Gastric liquid UB Kuujjuaq, QC BRS
GA0702E1CS 29 E3 2007 Clinical Gastric liquid UB Kuujjuaq, QC BRS
ME0702E1CS 29 E3 2007 Clinical Seal meat in oil UB Kuujjuaq, QC BRS
GA0808EPA 31 E3 2008 Clinical Gastric liquid UB Kuujjuaq, QC BRS
FE1010E1JL 32 E3 2010 Clinical Feces UB Kuujjuaq, QC BRS
GA1010E1JL 32 E3 2010 Clinical Gastric liquid UB Kuujjuaq, QC BRS
ME1010E1JL 32 E3 2010 Clinical Meat UB Kuujjuaq, QC BRS
GA1101E1BB 33 E10 2011 Clinical Gastric liquid UB Kuujjuaq, QC BRS
SE9908E E3 1999 Marine mammal Seal intestine UB Kuujjuaq, QC BRS
IN01SE63E1 E3 2001 Marine mammal Seal intestine UB Kuujjuaq, QC BRS (27)
MSKR5102E1 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (27)
MSKR5102E2 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (27)
PBKR-41E1 E10 2002 Environmental Peat bog UB Kuujjuaq, QC BRS (27)
FWSKR40E1 E10 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWSKR4802E1 E3 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWKR02E1 E3 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWKR11E1 E10 2004 Environmental Freshwater UB Kuujjuaq, QC BRS (27)
FWSK02-01E2 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWSK02-01E3 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWSK02-02E1 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWSK02-03E1 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWSK02-04E1 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWSK02-05E1 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWSK02-05E2 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWSK02-06E1 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWSK02-06E2 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWSK02-07E1 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWSK02-07E2 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWSK02-08E1 E10 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
FWSKR1302E1 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
RSKR-68E1 E3 2004 Environmental Coastal rock UB Kuujjuaq, QC BRS (27)
RSKR-68E2 E10 2004 Environmental Coastal rock UB Kuujjuaq, QC BRS (27)
RSKR-68E3 E3 2004 Environmental Coastal rock UB Kuujjuaq, QC BRS (27)
SO329E1 E11 2001 Environmental Shoreline soil UB Kuujjuaq, QC BRS (27, 37, 38)
SO329E2 E11 2001 Environmental Shoreline soil UB Kuujjuaq, QC BRS (27)
SOKR-14E1 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
SOKR-14E3 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
SOKR-18E1 E3 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
SOKR-19E1 E3 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS (27)
SOKR-20E1 E3 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
SOKR-22E1 E3 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
SOKR-22E3 E3 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
SOKR-23E1 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (27, 37, 38)
SOKR-23E3 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (27)
SOKR-24E1 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (27)
SOKR-24E2 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (27)
SOKR-24E3 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (27)
SOKR-25E2 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
SOKR-25E3 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
SOKR-27E1 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
SOKR-27E3 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
SOKR-29E1 E3 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS (27, 37, 38)
SOKR-29E2 E3 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS (27)
SOKR-33E1 E10 2002 Environmental Peat bog UB Kuujjuaq, QC BRS (27)
SOKR-34E1 E10 2002 Environmental Sediment UB Kuujjuaq, QC BRS (27)
SOKR-34E5 E10 2002 Environmental Sediment UB Kuujjuaq, QC BRS (27)
SOKR-35E1 E3 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS (27)
SOKR-35E3 E3 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS (27)
SOKR-37E1 E3 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (27)
SOKR-38E1 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (27)
SOKR-38E2 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (27)
SOKR-38E3 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (27)
SOKR-42E1 E10 2002 Environmental Shoreline soil UB Tasiujaq, QC BRS (27)
SOKR-43E1 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
SOKR-43E2 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
SOKR-44E1 E11 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS (27)
SOKR-44E2 E11 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS (27)
SOKR-44E3 E3 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS (27)
SOKR-46E1 E11 2004 Environmental Marine sediment UB Kuujjuaq, QC BRS (27)
SOKR-46E3 E3 2004 Environmental Marine sediment UB Kuujjuaq, QC BRS (27)
SOKR-49E1 E10 2002 Environmental Sediment UB Kuujjuaq, QC BRS (27)
SOKR-49E2 E10 2002 Environmental Sediment UB Kuujjuaq, QC BRS (27)
SOKR-50E1 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
SOKR-50E2 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
SP455–456E2 E11 2002 Environmental Coastal rock UB Kuujjuaq, QC BRS (27, 37, 38)
SP457–458E E3 2002 Environmental Coastal rock UB Kuujjuaq, QC BRS (27)
SW279E E3 2001 Environmental Seawater UB Kuujjuaq, QC BRS (27)
SW280E E11 2001 Environmental Seawater UB Kuujjuaq, QC BRS (27, 37, 38)
SWKR0402E1 E3 2004 Environmental Seawater UB Kuujjuaq, QC BRS (27)
SWKR0402E2 E3 2004 Environmental Seawater UB Kuujjuaq, QC BRS (27)
SWKR07E1 E3 2004 Environmental Seawater UB Kuujjuaq, QC BRS (27)
SWKR24E1 E11 2004 Environmental Seawater UB Kuujjuaq, QC BRS (27)
SWKR38E1 E10 2004 Environmental Seawater UB Kuujjuaq, QC BRS (27)
SWKR38E2 E10 2004 Environmental Seawater UB Kuujjuaq, QC BRS (27)
TRK02-02E1 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
TRK02-02E2 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
TRK02-03E2 E10 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
TRK02-04E1 E10 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
TRK02-04E3 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
TRK02-06E2 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
TRK02-06E3 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
TRK02-07E1 E3 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS (27)
TRK02-08E1 E10 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
TRK02-08E2 E11 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
TRK02-08E3 E10 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
TRK0208E3 E10 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (27)
FE9604ENT 11 E3 1996 Clinical Feces UB Quaqtaq, QC BRS
GA9604EAK 11 E3 1996 Clinical Gastric liquid UB Quaqtaq, QC BRS
GA9604ESM 11 E3 1996 Clinical Gastric liquid UB Quaqtaq, QC BRS
F9508EPB 9 E3 1995 Clinical Feces UB Tasiujaq, QC BRS (37, 38)
MI9608ESM 12 E3 1996 Clinical Seal meat UB Tasiujaq, QC BRS
GA9608EPB 13 E3 1996 Clinical Gastric liquid UB Tasiujaq, QC BRS
VI9608EPB 13 E3 1996 Clinical Seal meat UB Tasiujaq, QC BRS
GA9706EMA 16 E10 1997 Clinical Gastric liquid UB Tasiujaq, QC BRS
MI9706E 16 E3 1997 Clinical Igunaq UB Tasiujaq, QC BRS
GA0108EJC 25 E3 2001 Clinical Gastric liquid UB Tasiujaq, QC BRS
FE0201E1BC 26 E3 2002 Clinical Feces UB Tasiujaq, QC BRS
FE0202E1TC 26 E3 2002 Clinical Feces UB Tasiujaq, QC BRS
GA0202E1TS 26 E3 2002 Clinical Gastric liquid UB Tasiujaq, QC BRS
IG0201E2BC 26 E3 2002 Clinical Walrus igunaq UB Tasiujaq, QC BRS
IG0202E1 26 E3 2002 Clinical Walrus igunaq UB Tasiujaq, QC BRS
VO0202E1TC 26 E3 2002 Clinical Gastric liquid UB Tasiujaq, QC BRS
GL0410E1LC 28 E3 2004 Clinical Gastric liquid UB Tasiujaq, QC BRS
IG0410E2LC 28 E3 2004 Clinical Igunaq UB Tasiujaq, QC BRS
IG0410E3LC 28 E3 2004 Clinical Igunaq UB Tasiujaq, QC BRS
SO325E E3 2001 Environmental Shoreline soil UB Tasiujaq, QC BRS (27)
SO325E1 E3 2001 Environmental Shoreline soil UB Tasiujaq, QC BRS (27, 37, 38)
SO326E1 E3 2001 Environmental Shoreline soil UB Tasiujaq, QC BRS (27, 37, 38)
SOKR-3602E1 E3 2002 Environmental Shoreline soil UB Tasiujaq, QC BRS (27)
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a

Isolates (n = 175) were derived from samples collected during an outbreak (OB) investigation. Abbreviations: PAC, Pacific Coast; UKR, Ukraine; WHB, West Hudson Bay Coast; EHB, East Hudson Bay Coast; UB, Ungava Bay Coast; LAB, Labrador; BI, Baffin Island; QC, Quebec, Canada; ND, no details; BRS, Botulism Reference Service, Health Canada, Ottawa, Ontario, Canada; NML, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada. References are indicated (parentheses).

Mouse bioassay.

Mouse lethal dose (MLD) titers of botulinum neurotoxin were determined using 10-fold dilutions of culture supernatants in gelatin phosphate buffer according to standard methods (19).

bont coding sequence (CDS) determination and analysis.

SMALT v0.6.4 (http://www.sanger.ac.uk/resources/software/smalt/) was used to map reads to a genomic bont/E sequence (GenBank accession number EF028403) which included 2-kb flanking genomic region from Alaska E43 (NC_010723), and a Perl script using SamTools was used to obtain bont/E consensus CDS (filters: mapping q-score ≥ 20; base pair q-score ≥ 30; pileup ≥ 2) (20). Regions with gaps/low coverage were closed using PCR sequencing as described previously (1). Manual confirmation of variants was performed using IGV v2.0.24 (21) and Tablet v1.12.12.05 (22). Unique bont/E CDSs and translated bont/E amino acid sequences were identified using CD-Hit v4.6 (length/identity = 100%) (23). Illustrative multiple sequence alignments (see Fig. 2) were constructed in CLC Genomics Workbench v6.0.2.

FIG 2.

FIG 2

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CDS alignment depicting the distribution of nucleotide substitutions within bont/E1, bont/E3, and bont/E10 variants. Nucleotide substitutions (|), silent substitutions (×), and codon deletions (△) identified among bont/E variant CDSs indicated on the left (E1, italics; E3, regular font; E10, underlined) compared against reference sequences (E1, accession number X62683; E3, accession number EF028403; and E10, accession number KF861887) are shown.

Phylogeny analysis.

Maximum likelihood analysis of alignment files from ClustalW (v2.1) (24) was performed with RAxML (v7.2.8) (25) using a JTT+G substitution model for amino acids, and images were rendered in FigTree (v1.3.1) (http://tree.bio.ed.ac.uk/software/figtree/).

Nucleotide sequence accession numbers.

All bont CDSs (accession numbers in Table 2) identified in this study have been deposited in GenBank (http://www.ncbi.nlm.nih.gov/). Reference sequences for each bont/E gene variant were as follows: bont/E1, X62683; bont/E2, EF028404; bont/E3, EF028403; bont/E4, X62088; bont/E5, AB037711; bont/E6, AM695759; bont/E7, JN695729; bont/E8, JN695730; and bont/E9, JX424534. Unless otherwise specified, all accession numbers in this article are GenBank accession numbers.

TABLE 2.

bont/E CDS accession numbers of C. botulinum group II strains studied

Isolate bont/E CDS GenBank accession no. Variant type
E-RUSS KF861870 E1
E1 Dolman KF861868 E1
FE9709EBB2 KF861917 E10
FWKR11E1 KF861887 E10
FWSKR40E1 KF861906 E10
GA1101E1BB KF861907 E10
GA9706EMA KF861885 E10
MI19709E KF861914 E10
MI59709E KF861915 E10
MI69709E KF861919 E10
PBKR-41E1 KF861912 E10
SO301E KF861902 E10
SO303E1 KF861901 E10
SO303E3 KF861900 E10
SO303E4 KF861899 E10
SO303E5 KF861897 E10
SO304E1 KF861908 E10
SO304E2 KF861909 E10
SO305E1 KF861910 E10
SO307E1 KF861918 E10
SO309E2 KF861892 E10
SO322E1 KF861893 E10
SOKR-33E1 KF861891 E10
SOKR-34E1 KF878263 E10
SOKR-34E5 KF861895 E10
SOKR-49E1 KF861896 E10
SP417E-Alc KF861898 E10
SP417E-NT KF861894 E10
SWKR38E1 KF861888 E10
SWKR38E2 KF861889 E10
TRK02-03E2 KF861904 E10
TRK02-04E1 KF861905 E10
TRK02-08E1 KF861921 E10
TRK02-08E3 KF861922 E10
TRK0208E3 KF861886 E10
FE9709EKA KF861916 E10
SOKR-49E2 KF861890 E10
FWSK02-08E1 KF861903 E10
SO305E2 KF861911 E10
RSKR-68E2 KF861913 E10
SOKR-42E1 KF878264 E10
SO329E1 KF861875 E11
SO329E2 KF861882 E11
SOKR-44E1 KF861872 E11
SOKR-44E2 KF861874 E11
SOKR-46E1 KF861883 E11
SP455-456E2 KF861876 E11
SW280E KF861877 E11
SWKR24E1 KF861873 E11
TRK02-08E2 KF861871 E11
BE9708E1 KF719438 E3
Bennett KF719445 E3
CA9708E1 KF719439 E3
F9508EMA KF719431 E3
F9508EPB KF719430 E3
FE0005EJT KF719347 E3
FE0201E1BC KF719412 E3
FE0202E1TC KF719420 E3
FE0211E1DK KF719349 E3
FE0801E1IT KF719342 E3
FE1010E1JL KF719341 E3
FE9507EEA KF719443 E3
FE9604ENT KF719428 E3
FE9708E1JI KF719441 E3
FE9708E1PI KF719440 E3
FE9709EBB KF719328 E3
FE9709ECB KF719324 E3
FE9709ELB KF719329 E3
FE9908EDL KF719333 E3
FE9909ERG KF719330 E3
FWKR02E1 KF719350 E3
FWSK02-01E3 KF719386 E3
FWSK02-02E1 KF719403 E3
FWSK02-03E1 KF719401 E3
FWSK02–04E1 KF719361 E3
FWSK02-05E1 KF719365 E3
FWSK02-05E2 KF719366 E3
FWSK02-06E1 KF719415 E3
FWSK02-06E2 KF719409 E3
FWSK02-07E1 KF719408 E3
FWSK02-07E2 KF719407 E3
FWSKR1302E1 KF719404 E3
FWSKR4802E1 KF719336 E3
GA0108EJC KF719411 E3
GA0202E1TS KF719419 E3
GA0702E1CS KF719327 E3
GA0808EPA KF719343 E3
GA0811E1IT KF719346 E3
GA1010E1JL KF719339 E3
GA9604EAK KF719427 E3
GA9604ESM KF719426 E3
GA9608EPB KF719435 E3
GA9709EJA KF719326 E3
GA9709ENS KF719325 E3
GA9811E1MS KF719422 E3
GA9811E2MS KF719421 E3
Gordon KF719444 E3
IG0201E2BC KF719413 E3
IG0202E1 KF719417 E3
IG0410E2LC KF719348 E3
IG0410E3LC KF719410 E3
IN01SE63E1 KF719370 E3
INWB2202E1 KF719369 E3
ME0702E1CS KF719345 E3
ME1010E1JL KF719340 E3
MI9608ESM KF719437 E3
MI9706E KF719425 E3
MSKR5102E1 KF719406 E3
MSKR5102E2 KF719405 E3
MU0005EJT KF719331 E3
MU0103EMS KF719423 E3
MU8903E KF719322 E3
MU9708EJG-F235 KF719436 E3
RSKR-68E1 KF719334 E3
RSKR-68E3 KF719335 E3
S9510E KF719429 E3
SE9908E KF719372 E3
SO325E KF719376 E3
SO325E1 KF719394 E3
SO326E1 KF719395 E3
SOKR-14E1 KF719357 E3
SOKR-14E3 KF719356 E3
SOKR-18E1 KF719368 E3
SOKR-20E1 KF719362 E3
SOKR-22E1 KF719383 E3
SOKR-22E3 KF719384 E3
SOKR-23E1 KF719392 E3
SOKR-23E3 KF719397 E3
SOKR-24E1 KF719390 E3
SOKR-24E2 KF719402 E3
SOKR-24E3 KF719414 E3
SOKR-25E2 KF719400 E3
SOKR-25E3 KF719399 E3
SOKR-27E1 KF719323 E3
SOKR-27E3 KF719364 E3
SOKR-29E1 KF719389 E3
SOKR-29E2 KF719396 E3
SOKR-35E1 KF719373 E3
SOKR-35E3 KF719374 E3
SOKR-3602E1 KF719337 E3
SOKR-38E1 KF719355 E3
SOKR-38E2 KF719354 E3
SOKR-38E3 KF719353 E3
SOKR-43E1 KF719387 E3
SOKR-43E2 KF719380 E3
SOKR-44E3 KF719391 E3
SOKR-46E3 KF719351 E3
SOKR-50E1 KF719381 E3
SOKR-50E2 KF719382 E3
SW279E KF719367 E3
SWKR0402E1 KF719338 E3
SWKR0402E2 KF719393 E3
SWKR07E1 KF719332 E3
TRK02-02E1 KF719358 E3
TRK02-02E2 KF719359 E3
TRK02-04E3 KF719360 E3
TRK02-06E2 KF719377 E3
TRK02-06E3 KF719320 E3
V9804E KF719424 E3
VI9508E KF719432 E3
VI9608EPB KF719434 E3
VO0202E1TC KF719416 E3
GA0702E1 KF719352 E3
SO321E1 KF719398 E3
SOKR-37E1 KF719344 E3
MI9507E KF719433 E3
SP457-458E8 KF719375 E3
211 VH Dolman KF719447 E3
MU9708EJG-F236 KF719418 E3
GL0410E1LC KF719446 E3
FWSK02-01E2 KF719385 E3
INGR16-02E1 KF719371 E3
SOKR-19E1 KF719378 E3
TRK02-07E1 KF719379 E3
GA9709EHS KF878262 E3
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RESULTS

BoNT/E variant analysis.

The rate of food-borne botulism is notably high in certain northern communities (18, 26). Frequent, distinct botulism type E outbreaks in the Ungava Bay region have been reported (18), and a costal environmental sampling study has shown that C. botulinum spore counts can range from 270 to 1,800/kg in this area (27).

A total of 175 full-length bont/E CDSs were determined for clinical (n = 45), food (n = 21), marine mammal (n = 4), and environmental (n = 105) C. botulinum type E isolates from northern Canada (primarily from the banks of East Hudson Bay and Ungava Bay) (27), as well as isolates from the Pacific coast (211 VH Dolman, E1 Dolman) and the littoral of the Sea of Azov (E-RUSS) (Tables 1 and 2).

Sequence analysis of bont/E CDSs show clustering with previously described bont/E1 and bont/E3 variant types, as well as two distinct clades, termed E10 and E11; none of the other variants (E2 and E4 to E9) were represented. For the 175 type E isolates compared, 1.1% (n = 2) of the bont/E sequences cluster with E1, 71.4% (n = 125) with E3, 22.3% (n = 39) with E10, and 5.1% (n = 9) with E11 (Fig. 1).

FIG 1.

FIG 1

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Dendrogram comparing unique full-length bont/E CDSs from the current study to sequences of known bont/E variants (*) using RAxML (1,000 bootstraps). Accession numbers of the bont/E CDSs used for analysis are shown. Two clusters are distinct from bont/E variants E1 to E9 and are termed E10 and E11. The occurrence of each sequence is indicated (parentheses).

Within the bont/E3 cluster, nine unique CDSs were observed, the predominant one being identical to the publicly available sequence of strain Alaska E43 (accession number EF028403). Two bont/E3 CDSs (accession numbers KF719446 and KF719385) maintain the predicted amino acid sequence reported for Alaska E43, while five (accession numbers KF719418, KF719371, KF719378, KF719379, and KF878262) encode single amino acid substitutions and one (accession number KF719447) encodes two amino acid substitutions (Fig. 2 and data not shown). Amino acid variation within predicted BoNT/E3 variants is <0.2% (1 or 2 nucleotide substitutions; 0 to 2 amino acid substitutions). The variability does not appear localized to any domain.

Six different full-length CDS variants were identified in the bont/E10 cluster. The dominant form (corresponding to accession number KF861887) was highly represented (n = 33), while other forms were observed at a low frequency (Fig. 1). Predicted BoNT/E10 amino acid sequences varied by ≤0.56% (1 to 7 nucleotide substitutions; 1 to 7 amino acid substitutions) and are primarily localized to the receptor-binding domain (Fig. 2). A polymorphic site was also observed at position 3647/3648. No sequence variation was observed for bont/E CDSs in the E1 or E11 clusters.

At the amino acid level, the variant types E10 and E11 vary from all other E variants by 2.1 to 11.0%. The translated amino acid sequence of bont/E10 (accession number KF861887) resembles most closely variant E8, found present in an isolate from Lake Erie (2) (97.9%). When variant BoNT/E10 domains are considered separately, they are most similar to those of variant E8 (97.9%, 99.5%, and 96.4% identity for LC, HN, and HC, respectively). The overall translated amino acid sequence of bont/E11 is the most similar to BoNT/E10 (95.7% identity). At the level of the functional domains, BoNT/E11 domains are similar to those of BoNT/E10 (95.0% and 97.1% identical for LC and HC, respectively) and BoNT/E6 (95.2% identical for HN). A complete amino acid sequence identity comparison between BoNT variants E1 to E11 is presented in Table 3.

TABLE 3.

Nucleic acid and amino acid comparison of BoNT/E variantsa

Domain % identity
BoNT variant E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11
Holotoxin (1-3,756 nt/1-1,252 aa) E1 99.4 99.3 98.5 98.2 98.5 99.1 97.9 94.1 97.6 96.8
E2 99.1 98.7 98.2 97.9 98.3 98.5 98.5 94.0 97.9 97.1
E3 98.2 97.4 97.8 97.6 98.0 98.8 97.6 93.9 97.3 96.4
E4 97.3 97.0 95.6 97.1 98.3 98.1 97.9 94.5 97.4 96.6
E5 96.9 96.3 95.1 94.9 97.2 97.3 96.9 94.3 96.7 96.0
E6 97.0 96.8 95.9 96.9 94.8 98.2 98.3 93.7 97.8 96.8
E7 97.9 97.1 97.4 96.2 94.8 96.4 98.8 94.0 98.3 96.9
E8 96.2 97.0 95.7 96.1 94.1 96.8 98.3 94.0 99.0 97.4
E9 89.1 89.3 88.7 89.9 89.4 88.2 89.1 89.4 94.1 94.3
E10 95.4 95.8 94.7 94.8 93.4 95.6 96.8 97.9 89.4 97.9
E11 93.4 93.8 92.6 92.7 91.9 93.1 93.5 94.4 89.0 95.7
LC (1-1,260 nt/1-420 aa) E1 99.9 97.9 97.9 98.7 98.7 97.4 97.4 95.4 97.3 96.7
E2 99.8 97.9 97.8 99.9 98.6 97.4 97.4 95.3 97.2 96.6
E3 94.8 94.5 95.9 97.9 97.2 96.5 96.5 94.8 96.3 95.6
E4 96.0 95.7 91.0 97.9 96.8 96.8 96.8 96.5 96.6 96.0
E5 100.0 99.8 94.8 96.0 98.7 97.4 97.4 95.4 97.3 96.7
E6 96.7 96.4 93.3 93.6 96.7 97.9 97.9 94.7 97.6 96.3
E7 94.0 94.0 92.4 92.9 94.0 95.0 100.0 95.5 99.3 97.0
E8 94.0 94.0 92.4 92.9 94.0 95.0 100.0 95.5 99.3 97.0
E9 90.5 90.2 89.3 92.4 90.5 88.8 91.0 91.0 95.4 95.2
E10 93.8 93.6 91.9 92.1 93.8 94.3 97.9 97.9 90.7 97.7
E11 92.4 92.1 90.0 90.7 92.4 91.2 92.9 92.9 89.5 95.0
HN (1,261-2,499 nt/421-833 aa) E1 100.0 100.0 99.0 98.6 98.1 99.8 98.4 95.8 98.7 97.0
E2 100.0 100.0 99.0 98.6 98.1 99.8 98.4 95.8 98.7 97.0
E3 100.0 100.0 99.0 98.6 98.1 99.8 98.4 95.8 98.7 97.0
E4 98.3 98.3 98.3 98.5 98.4 98.8 98.8 96.4 99.1 97.4
E5 97.8 97.8 97.8 98.1 97.7 98.4 97.9 96.3 98.2 96.6
E6 97.1 97.1 97.1 97.8 96.9 98.1 98.9 95.6 99.1 97.6
E7 99.8 99.8 99.8 98.1 97.6 96.9 98.6 95.6 98.9 97.0
E8 97.3 97.3 97.3 98.1 97.1 98.3 97.6 95.6 99.7 97.3
E9 93.5 93.5 93.5 94.7 93.7 93.0 93.2 93.2 95.8 95.6
E10 97.8 97.8 97.8 98.6 97.6 98.8 98.1 99.5 93.7 97.5
E11 94.2 94.2 94.2 94.4 94.0 95.2 94.0 94.9 92.0 94.9
HC (2,500-3,756 nt/834-1,252 aa) E1 98.3 100.0 98.6 96.2 98.7 100.0 97.9 91.0 96.8 96.6
E2 97.6 98.3 98.0 95.2 98.1 98.3 99.6 90.9 97.8 97.7
E3 100.0 97.6 98.6 96.2 98.7 100.0 97.9 91.0 96.8 96.6
E4 97.6 97.1 97.6 95.1 99.6 98.6 98.1 90.6 96.6 96.4
E5 92.8 91.4 92.8 90.7 95.3 96.2 95.4 91.3 94.7 94.8
E6 97.4 96.9 97.4 99.3 90.9 98.7 98.2 91.0 96.7 96.6
E7 100.0 97.6 100.0 97.6 92.8 97.4 97.9 91.0 96.8 96.6
E8 97.4 99.8 97.4 97.4 91.2 97.1 97.4 91.0 98.2 97.9
E9 83.3 84.3 83.3 82.6 84.2 82.8 83.3 84.0 91.2 92.0
E10 94.5 96.2 94.5 93.8 88.8 93.8 94.5 96.4 83.8 98.5
E11 93.6 95.2 93.6 92.8 89.5 93.1 93.6 95.5 85.4 97.1
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a

Percent identity of nucleotide (boldface) and amino acid (lightface) sequences for bont/E CDS variants. Comparative values for the BoNT/E holotoxin and each domain (light chain [LC], translocation [HN], and receptor-binding [HC]) are presented. Strains (with sequences and accession numbers in parentheses) used for comparison are as follows: Beluga (E1, X62683), CDC5247 (E2, EF028404), Alaska E43 (E3, EF028403), BL5262 (E4, X62088), LCL155 (E5, AB037711), K35 (E6, AM695759), IBCA97-0192 (E7, JN695729), NYDH Bac-02-06430 (E8, JN695730), CDC66177 (E9, JX424534), FWKR11E1 (E10, KF861887), and SW280E (E11, KF861877). aa, amino acids.

Multiple-sequence alignments of variant bont/E CDSs were analyzed to examine the distribution of nucleotide substitution events (Fig. 3). The differences between bont/E10 and bont/E11 variant sequences and the other bont/E variant subtypes display a nonrandom substitution pattern. Extensive portions of the LC and HC are identical in bont/E10 and bont/E11 variants. Likewise, a significant region of the HN of bont/E10 was identical in all other bont/E variants except bont/E9 and bont/E11.

FIG 3.

FIG 3

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bont/E CDS alignment depicting the distribution of nucleotide substitutions among variants. The reference sequence used for each of the 11 alignments is indicated (left and Table 3), and horizontal tracks represent the alignment for each variant sequence (right). A scale bar denoting nucleotide position and catalytic light chain (LC), translocation (HN), and receptor binding (HC) domains is shown. The vertical lines plotted along the tracks indicate nucleotide substitutions (A, red; C, blue; G, yellow; T, green).

Geographic distribution of BoNT/E variants.

BoNT/E3 variant-carrying strains were isolated from outbreak-related clinical and/or food samples originating from British Columbia, the Northwest Territories, Nunavut (West Hudson Bay and Baffin Island), Labrador, and Quebec (Ungava Bay) (Fig. 4 and Table 1). BoNT/E3 environmental isolates were also obtained from the seaside along the Ungava Bay but were completely absent from the four East Hudson Bay shoreline sites surveyed. Only E10 isolates were identified from environmental samples collected from the eastern strand of East Hudson Bay. Although the Botulism Reference Service for Canada obtained positive results from clinical isolates related to a botulism case in this area in 1996, no isolates were recovered. E10 strains were also isolated from environmental samples from the Ungava Bay region where E10-related outbreaks were reported. The E11 variant was identified exclusively in environmental samples from the surroundings of the Koksoak River, a tributary of Ungava Bay, despite an extensive sampling effort along the Nunavik coastline. Moreover, no E11 strains were associated with a botulism outbreak.

FIG 4.

FIG 4

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Distribution of BoNT/E C. botulinum. The locations of origin for Canadian BoNT/E C. botulinum characterized in this study (Table 1) are shown. The BoNT/E variant type is indicated (legend, inset). Map source: Natural Resources Canada (http://atlas.gc.ca).

Toxicity of C. botulinum BoNT/E11-producing strains.

To determine if the apparent lack of an outbreak associated with E11 strains was related to their toxicity, 10-fold dilutions of culture supernatants from selected BoNT/E variants were assayed using the mouse bioassay. C. botulinum strains harboring BoNT/E1 (n = 2) and BoNT/E3 (n = 9) variants produced 103 to 105 and 102 to 105 mouse lethal doses (MLDs) per 0.5 ml of culture supernatant, respectively. BoNT/E11 (n = 7) and BoNT/E10 (n = 19) demonstrated reduced toxicity, with 102 to 104 and 101 to 103 MLDs per 0.5 ml (Table 4).

TABLE 4.

Mouse lethal doses of BoNT/E variants

Strain BoNT variant MLDa
E-RUSS E1 104–105
E1 Dolman E1 103–104
Bennett E3 104–105
Gordon E3 104–105
RSKR-68E1 E3 104–105
FE0005EJT E3 103–104
FE9708E1PI E3 103–104
GA9608EPB E3 103–104
MU8903E E3 103–104
INGR16-02E1 E3 103–104
FE9604ENT E3 102–103
SO329E1 E11 103–104
SOKR-44E1 E11 103–104
SW280E E11 103–104
SOKR-44E2 E11 102–103
SOKR-46E1 E11 102–103
SWKR24E1 E11 102–103
TRK02-08E2 E11 102–103
SO301E E10 102–103
SO303E5 E10 102–103
SO322E1 E10 102–103
SOKR-49E1 E10 102–103
SP417E-NT E10 102–103
SWKR38E1 E10 102–103
TRK02-04E1 E10 102–103
SO305E2 E10 102–103
RSKR-68E2 E10 102–103
FWKR11E1 E10 101–102
GA1101E1BB E10 101–102
MI69709E E10 101–102
SO303E1 E10 101–102
SO303E4 E10 101–102
SOKR-34E5 E10 101–102
SOKR-46E2 E10 101–102
SOKR-49E2 E10 101–102
TRK02-03E2 E10 101–102
FE9709EKA E10 101–102
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a

MLD per 0.5 ml of supernatant.

DISCUSSION

BoNT CDS variant determination was performed using phylogeny and further analyzed by nucleotide and amino acid identity analysis. E1 and E3, as well as two new toxin variants which we termed E10 and E11, were identified. Differences between newly identified bont/E10 and bont/E11 sequences and the other bont/E variants were not randomly distributed. For instance, the translocation domain of E10 is highly homologous to that found in all variants except E9 and E11, while large portions of its catalytic and receptor-binding domains are identical to those of E11. Such patterns suggest the occurrence of recombination events and have been previously reported for other bont genes, including bont/E variants, and toxin gene clusters (2, 9, 15, 28). The frequency of nucleotide substitutions observed within variants E10 (1 to 7 nucleotides) and E3 (1 or 2 nucleotides) is similar to that reported for variant B4 (1 to 5 nucleotides), a toxin variant generally associated with nonproteolytic C. botulinum (29).

Considering such a broad distribution, it is noteworthy that only 2 of the 175 strains included in our study (E1 Dolman, Pacific coast [30] E-RUSS, Sea of Azov [31]) encode BoNT/E1. E1 strains have been previously isolated in Washington State, Alaska, Labrador, Greenland, Denmark, Finland, and Japan (2, 12, 32). The vast majority of strains included in this study encode the BoNT/E3 variants, which were observed in environmental isolates from Ungava Bay as well as clinical and/or food isolates from British Columbia, the Northwest Territories, West Hudson Bay, Baffin Island, Labrador, and Ungava Bay. This widespread distribution throughout Canada is consistent with reports of E3 identification in samples from Alaska, the Great Lakes, Illinois, Finland, France, and Japan (2, 12, 14, 33). No E3 variants were identified among the 13 environmental isolates from four East Hudson Bay locales, which were all E10 variants. The latter were also observed in clinical and environmental samples from the Ungava Bay region. E11 isolates, however, were identified exclusively in environmental samples collected in the vicinity of the Koksoak River. The environmental survey focused on the Nunavik region (27); variants E10, E11, and other variant subtypes may be present in other areas of Canada.

Curiously, no outbreaks were associated with the BoNT/E11 variant. Intrinsic differences between C. botulinum type E strains, such as growth rate, toxin secretion, and toxin efficacy, may be responsible for the toxicity ranges observed. No game animals, to be used in the preparation of high-risk traditional aged-meat dishes, were butchered in the area where E11 strains were collected (18, 34). No high-risk traditional aged-meat dishes implicated in type E outbreaks from Kuujjuaq or the Ungava Bay area were found to be contaminated with E11 strains. BoNT/E3 strains were also found in the environment of the three butchering sites where BoNT/E11 were isolated (18, 34). The predominance and widespread distribution of other variants in the environment, in particular BoNT/E3, may explain the lack of human E11 cases reported to date. Likewise, no human botulism cases have been linked to strains harboring bont/E8 or bont/E9; these variants were identified in environmental isolates from the Great Lakes (2) and Dolavon, Chubut, Argentina, respectively (14). The identification and distribution of BoNT variants can provide insights during clinical investigations and can be applied to develop robust therapeutic and diagnostic technologies.

ACKNOWLEDGMENTS

This work was supported by Canadian Safety and Security Program project 07-219RD from Defense Research and Development Canada.

We thank Greg Sanders for technical assistance in performing mouse neutralization assays and members of the Genomics Core Services Group at the National Microbiology Laboratory (Winnipeg, Manitoba), headed by Morag Graham, for sequencing runs performed on the GAIIx instrument.

Footnotes

Published ahead of print 8 August 2014

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